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WO2010017083A1 - Procédés de traitement du cancer par des anticorps cd11b - Google Patents

Procédés de traitement du cancer par des anticorps cd11b Download PDF

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Publication number
WO2010017083A1
WO2010017083A1 PCT/US2009/052185 US2009052185W WO2010017083A1 WO 2010017083 A1 WO2010017083 A1 WO 2010017083A1 US 2009052185 W US2009052185 W US 2009052185W WO 2010017083 A1 WO2010017083 A1 WO 2010017083A1
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WIPO (PCT)
Prior art keywords
antibody
cancer
cdl
antibodies
cells
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PCT/US2009/052185
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English (en)
Inventor
Ghassan M. Saed
Zhongliang JIANG
Michael P. Diamond
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Wayne State University
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Publication of WO2010017083A1 publication Critical patent/WO2010017083A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2845—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta2-subunit-containing molecules, e.g. CD11, CD18

Definitions

  • Cancer is a disease that affects millions of people. Certain therapeutic antibodies are commercially available for the treatment of cancer. There is a need in the art for additional methods and compositions which employ antibodies for treating cancer.
  • the present invention relates to methods of treating a subject suffering from a cancer comprising: administering, to a subject suffering from a cancer, a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an anti-CD 1 Ib antibody.
  • the anti-CD 1 Ib antibody competes for binding with antibody WT.5.
  • the antibody is antibody WT.5.
  • the antibody induces apoptosis in SKOV, MDAH- 2774, or BxPC-3 cells.
  • the antibody induces caspase-3 activity in SKOV, MDAH-2774, or BxPC-3 cells.
  • a pharmaceutical composition may be administered intravenously.
  • the antibody is a monoclonal CDl Ib antibody.
  • the CDl Ib antibody is an anti-human CDl Ib antibody.
  • the antibody is an IgG antibody.
  • the antibody is an IgA antibody.
  • An antibody of the present invention may be administering with one or more agents selected from the group consisting of: gemcitabine, paclitaxel, imatinib mesylate, sunitinib malate, adriamycin, daunomycin, cisplatin, etoposide, vinblastine, vincristine, and methotrexate.
  • the cancer is selected from the group consisting of: uterine cancer, bowel cancer, adenocarcinoma, cancer of the fallopian tube, cancer of the cervix, pancreatic cancer, ovarian cancer, endometrial cancer, liver cancer, prostate cancer, lung cancer, and breast cancer.
  • the cancer is a diffuse large cell lymphoma.
  • the present invention relates to treating or preventing cancer by administering a CDl Ib antigen.
  • a physician may assess whether a patient is suffering from a cancer by standard clinical indices, including radiological methods (e.g., x-rays), surgical procedures, and other diagnostic procedures known in the art.
  • radiological methods e.g., x-rays
  • surgical procedures e.g., surgical procedures, and other diagnostic procedures known in the art.
  • antibody refers to a monomeric (e.g., single chain antibodies) or multimeric polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • antibody also includes antigen-binding polypeptides such as Fab, Fab', F(ab') 2 , Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, and diabodies.
  • CDR complementarity determining region
  • the term antibody includes polyclonal antibodies and monoclonal antibodies unless otherwise indicated.
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
  • the amino terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.
  • a Fd fragment means an antibody fragment that consists of the V n and C n 1 domains; an Fv fragment consists of the V L and V n domains of a single arm of an antibody; and a dAb fragment (Ward et al., Nature 341:544-546 (1989)) consists of a V n domain.
  • the antibody is a single-chain antibody (scFv) in which a V L and V n domains are paired to form a monovalent molecule via a synthetic linker that enables them to be made as a single protein chain.
  • scFv single-chain antibody
  • the antibodies are diabodies, i.e., are bivalent antibodies in which V n and V L domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites.
  • a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites.
  • an "anti-CD 1 Ib” antibody is an antibody that specifically binds a CDl Ib polypeptide.
  • CDl Ib polypeptides include, but are not limited to, a mouse CDl Ib polypeptide (e.g., SEQ ID NO: 1), a rat CDl Ib polypeptide (e.g., SEQ ID NO: 3), and a human CDl Ib polypeptide (e.g., SEQ ID NO: 3).
  • An example of an "anti-CD 1 Ib antibody” is antibody WT.5 a mouse anti-rat IgA monoclonal antibody to rat CDl Ib (see Tamatani et al. (1993) Eur. J. Immunol.
  • immunoassay is an assay that uses an antibody to specifically bind an antigen.
  • the immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
  • an antibody specifically binds an antigen when it has a IQ of at least about 1 ⁇ M or lower, more usually at least about 0.1 ⁇ M or lower, and preferably at least about 10 nM or lower for that antigen.
  • a variety of immunoassay formats e.g., Western blots, ELISAs, etc. may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, (1990) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • human antibody means any antibody in which the variable and constant domain sequences are human sequences.
  • the term encompasses antibodies with sequences derived from human genes, but which have been changed, e.g. to decrease possible immunogenicity, increase affinity, eliminate cysteines that might cause undesirable folding, etc.
  • the term encompasses such antibodies produced recombinantly in non-human cells, which might impart glycosylation not typical of human cells. These antibodies may be prepared in a variety of ways, as described below.
  • chimeric antibody as used herein means an antibody that comprises regions from two or more different antibodies.
  • one or more of the CDRs are derived from a human anti-CD 1 Ib antibody.
  • all of the CDRs are derived from a human anti-CD 1 Ib antibody.
  • the CDRs from more than one human anti-CD 1 Ib antibodies are combined in a chimeric antibody.
  • a chimeric antibody may comprise a CDRl from the light chain of a first human anti-CD 1 Ib antibody, a CDR2 from the light chain of a second human anti-CD 1 Ib antibody and a CDR3 from the light chain of a third human anti-CD l ib antibody, and the CDRs from the heavy chain may be derived from one or more other anti-CD 1 Ib antibodies.
  • the framework regions may be derived from one of the anti-CD 1 Ib antibodies from which one or more of the CDRs are taken or from one or more different human antibodies.
  • one or more CDRs from a non-human species (e.g., mouse or rat) antibody may be recombinantly inserted into a human antibody framework resulting in a "humanized" antibody.
  • FIG. 1 depicts a bar graph of caspase-3 activity, which was measured in the human epithelial ovarian cancer cell lines SKOV and MDAH2774, and the pancreatic cancer cell line BxPC-3 treated without and with 50 ⁇ g/ml of a mouse anti-rat CDl Ib antibody (BD Biosciences, CA), as described in Example 1.
  • FIG. 2 depicts immunofluorescence staining with FITC-conjugated antibody (mouse anti-CD 1 Ib monoclonal antibody, Santa Cruz Biotechnology Inc., Santa Cruz, CA) diluted at 1 : 100 ratio for 1 hour at room temperature as as described in Example 2 for various cancer tissues and their normal counterparts.
  • FIG. 1 depicts a bar graph of caspase-3 activity, which was measured in the human epithelial ovarian cancer cell lines SKOV and MDAH2774, and the pancreatic cancer cell line BxPC-3 treated without and with 50 ⁇ g/ml of a mouse anti-rat CDl Ib antibody (BD Biosciences
  • FIG. 3 depicts the results of a time course of viability of WSU-DLCL2 cells incubated in the absence (T) or presence of a CD-I Ib antibody (A).
  • the x- axis is the incubation time in hours with or without the CD-I Ib antibody.
  • the y- axis represents the number of viable cells present (xlO 5 ) The cells were assayed for viability using a trypan blue assay.
  • the present invention relates to methods of treating a subject suffering from cancer by administering a therapeutically effective amount of an anti-CDl Ib antibody.
  • Methods have been described for generating CDl Ib antibodies (Tamatani et al. (1993) Eur. J. Immunol. 23(9):2181-2188).
  • the cancer can be treated prophylactically, acutely, and chronically using antibodies of the present invention, depending on the nature of the disease.
  • CD l ib monoclonal or polyclonal antibodies For preparation of CD l ib monoclonal or polyclonal antibodies, techniques known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985)).
  • phage display technology can be used to identify single chain antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al, Nature 348:552-554 (1990); Marks et al, Biotechnology 10:779-783 (1992)).
  • CDl Ib polypeptides, wild-type cells (e.g., neutrophils), or cells expressing recombinant CDl Ib polypeptides are employed as antigens to generate CDl Ib antibodies.
  • synthetic peptides derived from CDl Ib amino sequences disclosed herein and conjugated to a carrier protein can be used as an immunogen.
  • recombinant CDl Ib polypeptides can be employed to generate cognate antibodies.
  • recombinant mouse CDl Ib e.g., SEQ ID NO: 1
  • rat CDl Ib e.g., SEQ ID NO: 2
  • human CDl Ib e.g., SEQ ID NO: 3
  • nucleic acids encoding CDl Ib can be made or isolated using routine techniques in the field of recombinant genetics and synthetic nucleic acid chemistry.
  • Polyclonal antibodies typically can be generated by immunization of an animal with the antigen of choice.
  • the immunization of the animals can be by any method known in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990.
  • mice rabbits, rats, sheep, goats, pigs, cattle and horses are well known in the art. See, e.g., Harlow and Lane, supra, and U.S. Patent 5,994,619.
  • a CDl Ib antigen is administered with an adjuvant to stimulate the immune response.
  • adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes).
  • RIBI muramyl dipeptides
  • ISCOM immunonostimulating complexes
  • polyclonal antibodies and/or antibody -producing cells can be obtained from the animal.
  • anti-CD 1 Ib antibody-containing serum is obtained from the animal by bleeding or sacrificing the animal.
  • the serum may be used as it is obtained from the animal, an immunoglobulin fraction may be obtained from the serum, or the anti-CD 1 Ib antibodies may be purified from the serum.
  • the animal's immune response to an immunogen preparation can be monitored by taking test bleeds and determining the titer of reactivity to the protein of choice. When appropriately high titers of antibody to the immunogen are obtained, blood can be collected from the animal and antisera are prepared.
  • the level of CDl Ib antibodies in serum can be assayed using a CDl Ib immunoassay.
  • the polyclonal antibodies can be purified from the serum of an immunized animal using standard antibody and protein purification techniques.
  • Monoclonal antibodies can also be prepared against CDl Ib.
  • hybridoma techniques can be used to generate monoclonal antibodies.
  • antibody-producing immortalized cell lines can be prepared from cells isolated from the immunized animal. After immunization, the animal is sacrificed and lymph node and/or splenic B cells are immortalized.
  • Methods of immortalizing cells include, but are not limited to, transfecting them with oncogenes, infecting them with an oncogenic virus, cultivating them under conditions that select for immortalized cells, subjecting them to carcinogenic or mutating compounds, fusing them with an immortalized cell, e.g., a myeloma cell, and inactivating a tumor suppressor gene. See, e.g., Harlow and Lane, supra. If fusion with myeloma cells is used, the myeloma cells preferably do not secrete immunoglobulin polypeptides (a non-secretory cell line).
  • Immortalized cells can be screened using CD 1 Ib polypeptides, or portions thereof, or a cell expressing CDl Ib.
  • the initial screening can be performed using an enzyme-linked immunoassay (ELISA) or a radioimmunoassay.
  • ELISA enzyme-linked immunoassay
  • radioimmunoassay a radioimmunoassay
  • human antibodies are produced by immunizing a non-human animal comprising in its genome some or all of human immunoglobulin heavy chain and light chain loci with a CDl Ib antigen.
  • the non-human animal can be a XENOMOUSETM animal (Abgenix Inc., Fremont, CA).
  • HuMAb- Mouse® a transgenic mouse produced by Medarex (Medarex, Inc., Princeton, NJ).
  • XENOMOUSETM mice are engineered mouse strains that comprise large fragments of human immunoglobulin heavy chain and light chain loci and are deficient in mouse antibody production. See, e.g., Green et ah, Nature Genetics 7: 13-21 (1994) and U.S. Patents 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598, 6,130,364, 6,162,963 and 6,150,584.
  • the splenic B cells from a XENOMOUSETM can be fused to a non-secretory mouse myeloma (e.g, the myeloma cell line P3-X63-AG8-653) and monoclonal antibodies may be identified from the resulting pool of hybridomas.
  • the CDl Ib antibodies secreted by a hybridoma may be purified from a hybridoma culture and used in the methods of the present invention.
  • the nucleic acids encoding the heavy and light chains of the CDl Ib antibody may be isolated from a hybridoma and expressed in a host cell, e.g., NSO cells, CHO cells etc., to provide a source material from which purified CDl Ib antibodies may be obtained.
  • a transgenic animal is immunized with CDl Ib, primary cells, e.g., spleen or peripheral blood cells, are isolated from an immunized transgenic animal and individual cells producing antibodies specific for the desired antigen are identified.
  • Polyadenylated mRNA from each individual cell is isolated and reverse transcription polymerase chain reaction (RT-PCR) is performed using sense primers that anneal to variable region sequences, e.g., degenerate primers that recognize most or all of the FRl regions of human heavy and light chain variable region genes and antisense primers that anneal to constant or joining region sequences.
  • RT-PCR reverse transcription polymerase chain reaction
  • the cDNAs of the heavy and light chain variable regions are then cloned and expressed in any suitable host cell, e.g., a myeloma cell, as chimeric antibodies with respective immunoglobulin constant regions, such as the heavy chain and K or ⁇ constant domains.
  • a suitable host cell e.g., a myeloma cell
  • immunoglobulin constant regions such as the heavy chain and K or ⁇ constant domains.
  • Anti CDl Ib antibodies may then be identified and isolated as described herein.
  • the invention provides a method for making humanized anti-CD 1 Ib antibodies.
  • rats or mice are immunized with a CDl Ib antigen as described below under conditions that permit antibody production.
  • Antibody-producing cells are isolated from the animals, fused with myelomas to produce hybridomas, and nucleic acids encoding the heavy and light chains of an anti-CD 1 Ib antibody of interest are isolated. These nucleic acids are subsequently engineered using techniques known to those of skill in the art and as described further below to reduce the amount of non-human sequence, i.e., to humanize the antibody to reduce the immune response in humans [0029]
  • phage display techniques can be used to provide libraries containing a repertoire of antibodies with varying affinities for CDl Ib.
  • one method for preparing the library of antibodies for use in phage display techniques comprises the steps of immunizing a non- human animal comprising human immunoglobulin loci with a CDl Ib polypeptide to create an immune response, extracting antibody producing cells from the immunized animal; isolating RNA from the extracted cells, reverse transcribing the RNA to produce cDNA, amplifying the cDNA using a primer, and inserting the cDNA into a phage display vector such that antibodies are expressed on the phage.
  • the resulting phage are tested for immunoreactivity to a CDl Ib polypeptide.
  • Recombinant anti-CD 1 Ib antibodies of the invention may be obtained in this way.
  • kits for generating phage display libraries e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the Stratagene SurfZAP TM phage display kit, catalog no. 240612
  • commercially available systems for producing fully human phage expressed antibodies such as Cambridge Antibody Technology PLC (Cambridge, United Kingdom) and MorphoSys AG (e.g., HuCAL® GOLD technology, Martinsried, Germany).
  • nucleic acids encoding the selected antibody can be recovered from the display package (e.g., from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques.
  • the DNA encoding a phage expressed antibody can be cloned into a recombinant expression vector and introduced into a mammalian host cells or prokaryotic cells as appropriate for that antibody.
  • a CDl Ib antibody of the present invention competes for binding to a CDl Ib polypeptide, a fragment of a CDl Ib polypeptide, or a cell that expresses CDl Ib.
  • the amount of competition can be determined in a competitive binding assay in which a test antibody inhibits specific binding of a reference antibody (e.g., antibody WT.5) to a given CDl Ib target.
  • a reference antibody e.g., antibody WT.5
  • Examples of competitive binding assays are known in the art (see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press (1988)).
  • such an assay involves the use of a CDl Ib antigen, an unlabelled test antibody and a labeled reference antibody.
  • CDl Ib antibodies identified in a competitive binding assay that are able to compete for the binding to a CDl Ib antigen with the labeled antibody include CDl Ib antibodies that bind to the same epitope as the reference CDl Ib antibody and CDl Ib antibodies that bind to another eptiope that is sterically hindered by the binding of the reference CDl Ib antibody.
  • the specific binding of a competing test CDl Ib antibody to a CDl 1 antigen typically inhibits the binding of the CDl Ib test antibody by 50%, when the CDl Ib reference antibody is in excess to the CDl Ib test antibody.
  • the test antibody can by labeled with a detectable label such a radioactive label, a fluorescent label, a biotinylated label, etc.
  • a CDl Ib antibody of the present invention induces apoptosis in SKOV, MDAH-2774, or BxPC-3 cells.
  • Apoptosis may be measured using methods known in the art such as terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, DNA fragmentation assays, and caspase-3 assays.
  • TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling
  • the invention also relates to pharmaceutical compositions comprising an anti-CD 1 Ib antibody for the treatment of subjects in need of treatment for cancer.
  • Treatment may involve administration of one or more anti-CD 1 Ib monoclonal antibodies of the invention, alone or with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride can be present in the composition.
  • additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody.
  • compositions of this invention may be in a variety of forms, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.
  • liquid solutions e.g., injectable and infusible solutions
  • dispersions or suspensions tablets, pills, powders, liposomes and suppositories.
  • Typical compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans.
  • Therapeutic compositions typically are sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration.
  • Sterile injectable solutions can be prepared by incorporating the anti-CD 1 Ib antibody in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the methods of preparation include vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • the antibody composition may be prepared with a carrier that will protect the antibody against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • the present invention provides for methods for treating a subject suffering from cancer by administering a therapeutically effective amount of an anti-CD 1 Ib antibody to a subject in need thereof.
  • a “therapeutically effective amount” refers to an amount, at dosages and for periods of time necessary, sufficient to inhibit, halt, or allow an improvement in the disorder or condition being treated when administered alone or in conjunction with another pharmaceutical agent or treatment in a particular subject or subject population.
  • subject refers to a member of the class Mammalia. Examples of mammals include, without limitation, humans, primates, chimpanzees, rodents, mice, rats, rabbits, horses, dogs, cats, sheep, and cows.
  • a therapeutically effective amount can be determined experimentally in a laboratory or clinical setting, or may be the amount required by the guidelines of the United States Food and Drug Administration, or equivalent foreign agency, for the particular disease and subject being treated. [0041] It should be appreciated that the determination of proper dosage forms, dosage amounts, and routes of administration is within the level of ordinary skill in the pharmaceutical and medical arts.
  • a therapeutically effective amount of the antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of an agent are outweighed by the therapeutically beneficial effects.
  • the antibody may be administered once or multiple times.
  • the antibody may be administered from three times daily to once every six months or longer.
  • the administering may be on a schedule such as three times daily, twice daily, once daily, once every two days, once every three days, once weekly, once every two weeks, once every month, once every two months, once every three months and once every six months.
  • Co-administration of an antibody with an additional therapeutic agent encompasses administering a pharmaceutical composition comprising the anti-CD 1 Ib antibody and the additional therapeutic agent and administering two or more separate pharmaceutical compositions, one comprising the anti-CD 1 Ib antibody and the other(s) comprising the additional therapeutic agent(s).
  • co-administration or combination therapy refers to antibody and additional therapeutic agents being administered at the same time as one another, as wells as instances in which an antibody and additional therapeutic agents are administered at different times. For instance, an antibody may be administered once every three days, while the additional therapeutic agent is administered once daily. Alternatively, an antibody may be administered prior to or subsequent to treatment of the disorder with the additional therapeutic agent. An antibody and one or more additional therapeutic agents (the combination therapy) may be administered once, twice or at least the period of time until the condition is treated, palliated or cured.
  • the CDl Ib antibodies of the present invention may be co-administered with agents that are useful for the treatment of cancer (e.g., drugs such as gemcitabine, paclitaxel, imatinib mesylate, sunitinib malate, adriamycin, daunomycin, cisplatin, etoposide, vinblastine, vincristine, and methotrexate). Radiation treatments can also be co-administered with a compound of the present invention for the treatment of cancers.
  • therapeutic antibodies such as AVASTIN ® can be co-administered with a CDl Ib antibody.
  • biologies such as small interfering RNAs (siRNAs), antisense molecules, growth factors, cytokines, etc. may be co-administered with CDl Ib antibodies of the present invention.
  • the antibodies of the present invention can be administered by a variety of methods known in the art including, via an intraarterial, intravenous, subcutaneous, intramuscular, or parenteral routes.
  • the antibody is administered by intravenous infusion or injection.
  • the route and/or mode of administration will vary depending upon the desired results.
  • Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • Parenteral compositions can be formulated in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier [0047]
  • An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the invention from 1 to 40 mg/kg.
  • the dose is 1-500 mg. In other embodiments, the dose is 50-300 mg. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. [0048] In further embodiments, the present invention relates to the use of CDl Ib antigens as a cancer vaccine. In particular embodiments, the present invention relates to administering a CDl Ib antigen to prevent or treat cancer.
  • CD l ib antibodies of the present invention may be tested in vivo in animal models of cancer.
  • the CDl Ib antibodies may be tested using a xenograft model in Severe Combined Immunodeficiency (SCID) mice.
  • SCID Severe Combined Immunodeficiency
  • a CDl Ib antibody can be tested against pancreatic cancer in vivo using a xenograft models of human pancreatic cancer as follows: [0100] 1) The Maximum Tolerated Dose (MTD) of the CDl Ib antibody can be determined.
  • MTD Maximum Tolerated Dose
  • the MTD of a CD 1 Ib antibody in SCID mice can be determined.
  • the MTD of a CD l ib antibody is defined as the dose that will lead to no death of any of the animals and no more that 10% loss of body weight within first two weeks followed by weight gain.
  • a CDl Ib antibody (e.g., antibody WT.5) can be prepared in PBS and administered to SCID mice at the desired final dose. MTD studies can be done using four-week-old SCID, which can be obtained from Taconic Laboratory (Germantown, N.Y.). After a few days of adaptation, 10 animals would be used for each dose level and their initial weights are recorded. The CDl Ib antibody would be given intravenously (IV) via a tail vein. The animals would be weighed daily and monitored for activity, skin changes indicating dehydration (secondary to diarrhea) and any other physical or behavioral abnormalities. [0056] Subject to suitable adjustment, a representative procedure for the MTD determination is described as follows:
  • a dose-range finding study of at least three levels of the CDl Ib antibody candidate plus a vehicle-only control groups given drug intravenously (IV) weekly for 3 consecutive weeks would be conducted in SCID mice. There would be 10 animals per group. Animal survival would be monitored for an additional 3 weeks.
  • the MTD is defined as the dose that would lead to no deaths of any of the animals and no more than 10% loss of body weight during treatment, followed by weight gain. MTD studies would be done on non-tumor bearing SCID mice.
  • [0058] 2 Determination of the anti-tumor activity of CDlIb antibody given IV and orally using human pancreatic cancer xenograft models BXPC3-SCID as a subcutaneous-model.
  • Tumor weight (mg) (A X B2)/2 where A and B are the tumor length and width (in mm), respectively.
  • Tumor growth inhibition (T/C) is the median tumor weight in the treated group (T) when the median tumor weight in the control group reach 750 mg. Results are expressed as percentage. According to NCI-accepted criteria, a treatment is considered effective if T/C is ⁇ 42%.
  • Tumor growth delay (T-C) is the difference between the median time (in days) required for the treatment group tumors (T) to reach 750 mg and the median time (days) for the control group tumors to reach the same weight.
  • the human epithelial ovarian cancer cell lines MDAH-2774 and SKO V-3 were obtained from American Type Culture Collection (ATCC) (Manassas, VA). Cell lines were cultured in 75cm 2 cell culture flask (Corning Incorporated, Corning, NY) with McCoy's 5 A medium (Invitrogen, CA) supplemented with 10% fetal bovine serum (FBS) (ATCC). Culture medium was replaced every two days.
  • the human pancreatic cell line BxPC-3 was a kind gift from Dr. Ramzi Muhammed, Department of Hematology and Oncology, Wayne State University, Detroit, Michigan. BxPC-3 cells are also available from American Type Culture Collection (ATCC) (Manassas, VA).
  • BxPC-3 was cultured in RPMI medium 1640 (Invitrogen, CA). For each experiment, cells were plated in 60 mm x 15 mm cell culture dishes at a cell density of approximately 2 X 10 6 cells per dish and cultured for another 24 hours with and without 50 ⁇ g/ml of media of a mouse anti-rat CDl Ib ((Integrin ⁇ M chain, Mac-1 ⁇ chain) IgA (K) monoclonal antibody (Cat. No. 554980, BD Biosciences, CA).
  • a mouse anti-rat CDl Ib ((Integrin ⁇ M chain, Mac-1 ⁇ chain) IgA (K) monoclonal antibody
  • This antibody also known as WT.5 antibody, reacts with the ⁇ subunit of Mac-1 ( ⁇ M ⁇ 2 , CDl lb/CD18) found on neutrophils and myeloid cells, but not on lymphocytes (see Tamatani et al. (1993) Eur. J. Immunol. 23(9):2181-2188; Watanabe et al. (1997) Am. J. Pathol. 150(3):971-979). All experiments were performed in triplicate. Example 1 Caspase-3 activity.
  • Chemicon's Caspase-3 Colorimetric Activity Assay Kit (Chemicon International, CA) was used which provides a simple and convenient means for assaying the activity of caspases that recognize the sequence DEVD (Asp-Glu- VaI- Asp).
  • the assay is based on spectophotometric detection of the chromophore p-nitroaniline (pNA) after cleavage from the labeled substrate DEVD-pNA.
  • the free pNA can be quantified using a spectrophotometer or a microtiter plate reader at 405 nm. Comparison of the absorbance of pNA from a CDl Ib antibody treated sample with an untreated control allows determination of the fold increase in caspase-3 activity.
  • Cells (2 x 10 6 ) were harvested and lysed in 300 ⁇ l of cell lysis buffer included with the kit, and concentrations were equalized for each sample set. Subsequently, 150 ⁇ g of cell lysate was combined with substrate reaction buffer containing 30 ⁇ g of caspase-3 substrate, acetyl-DEVD-p-nitroaniline (Ac- DEVD-pNA). This mixture was incubated for 1 hour at 37°C, and then absorbance was measured with a plate reader (Ultramark, BIO-RAD, Hercules, CA) by detection of the chromophore p-nitroaniline (pNA) after cleavage from the labeled substrate DEVD-pNA.
  • a plate reader Ultramark, BIO-RAD, Hercules, CA
  • the amount of pNA ( ⁇ M) is graphed for the respective treatment in Figure 1.
  • the cells treated with the CDl Ib antibody had significantly higher caspase-3 activity than the cells that were not treated with the CDl Ib antibody.
  • Tissue sections For tissue sections embedded in paraffin and mounted on a slide were heated in an oven at 65°C for 24 hours up in order to help the tissue adhere to the slide. Tissue sections were deparaffinize by immersing them in xylenes 2 times, 5 minutes each at room temperature. Tissues were hydrated with 100%, 95%, and 80% ethanol 2 times, 3 minutes each at room temperature. Slides were washed in 0.05% Tween-20 diluted in PBS 3 times, 5 minutes each. Endogenous peroxidase activity was blocked by submerging the slide in 3% hydrogen peroxide for 20 minutes, followed by washing in 0.05% Tween-20 diluted in PBS 3 times, 5 minutes each.
  • Antigen retrieval was done by submerging slides in the pre-heated citrate buffer (95-100 0 C) in a water bath for 30 minutes. Sections were blocked with the 5 % bovine serum albumin (BSA) diluted in PBS at room temperature in the dark.
  • BSA bovine serum albumin
  • WSU-DLCL2 cells (a human diffuse large cell lymphoma cell line) were seeded at 2 x 10 5 cells/ml in a 24 well plate, then were untreated, or CD-I 1 antibody treated (20 ⁇ g/ml) (mouse anti-rat CDl Ib (Integrin ⁇ M chain, Mac-1 ⁇ chain) IgA (K) monoclonal antibody (Cat. No. 554980, BD Biosciences, CA) in duplicates. Cells were counted every 24 hours for a 72 hours incubation period using trypan blue exclusion assay (sigma) (Mohammad RM, Wang S, Aboukameel A, Chen B, Wu X, Chen J, Al-Katib A.

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Abstract

L'invention porte sur des procédés et sur des compositions pour traiter un cancer par des anticorps CD11b. Les anticorps peuvent être des anticorps WT.5 ou entrer en compétition avec des anticorps WT.5, et induire l'apoptose dans des cellules SKOV, MDAH-2774 ou BxPC-3.
PCT/US2009/052185 2008-08-04 2009-07-30 Procédés de traitement du cancer par des anticorps cd11b WO2010017083A1 (fr)

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WO2016197974A1 (fr) * 2015-06-12 2016-12-15 Yen-Ta Lu Méthodes et anticorps pour moduler une réponse immunitaire

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2649995A2 (fr) * 2008-02-29 2013-10-16 The Board Of Trustees Of The Leland Stanford Junior University Inhibition de la croissance tumorale post-radiation
EP2649995A3 (fr) * 2008-02-29 2014-05-21 The Board Of Trustees Of The Leland Stanford Junior University Inhibition de la croissance tumorale post-radiation
WO2016197974A1 (fr) * 2015-06-12 2016-12-15 Yen-Ta Lu Méthodes et anticorps pour moduler une réponse immunitaire
CN107949571A (zh) * 2015-06-12 2018-04-20 台湾基督长老教会马偕医疗财团法人马偕纪念医院 调控免疫反应的方法及抗体
JP2018524300A (ja) * 2015-06-12 2018-08-30 マッカイ メディカル ファンデーション ザ プレスビュテロス チャーチ イン タイワン マッカイ メモリアル ホスピタル 免疫応答を調節するための方法および抗体
AU2016276509B2 (en) * 2015-06-12 2021-04-01 Mackay Medical Foundation The Presbyterian Church In Taiwan Mackay Memorial Hospital Methods and antibodies for modulation of immunoresponse
RU2757489C2 (ru) * 2015-06-12 2021-10-18 Маккэй Медикл Фаундэйшн Зе Пресбайтериэн Чёч Ин Тайвэнь Маккэй Мемориал Хоспитэл Способы и антитела для модуляции иммунного ответа
JP7284557B2 (ja) 2015-06-12 2023-05-31 マッカイ メディカル ファンデーション ザ プレスビュテロス チャーチ イン タイワン マッカイ メモリアル ホスピタル 医薬組成物
KR20230091184A (ko) * 2015-06-12 2023-06-22 매카이 메디컬 파운데이션 더 프레즈비티리언 처치 인 타이완 매카이 메모리얼 호스피탈 면역반응의 조절을 위한 방법 및 항체
US11685785B2 (en) 2015-06-12 2023-06-27 Ascendo Biotechnology, Inc. Methods and antibodies for modulation of immunoresponse
KR102661066B1 (ko) 2015-06-12 2024-04-25 매카이 메디컬 파운데이션 더 프레즈비티리언 처치 인 타이완 매카이 메모리얼 호스피탈 면역반응의 조절을 위한 방법 및 항체

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